Two human proteins, p21 and p27, bind to and regulate cyclin-dependent kinases (Cdks), the master time- keepers of eukaryotic cell division. p21 and p27 are intrinsically unstructured proteins. Importantly, the intrinsic flexibility of p27 mediates sequential folding upon binding to Cdk/cyclin complexes. While lacking tertiary structure, p27 exhibits partially folded local structural elements prior to binding Cdk/cyclin complexes, which influences the sequential binding mechanism. The PI's group recently elucidated a two-step phosphorylation mechanism that relieves p27-mediated inhibition of Cdk2 through tyrosine (step 1) and threonine (step 2) phosphorylation of p27. After phosphorylation on tyrosine 88, Cdk2 itself phosphorylates p27 on threonine 187, targeting it for ubiquitination and degradation. Tyrosine kinases that promote cell proliferation were shown to initiate this two-step mechanism that eliminates p27-mediated cell cycle arrest at the G1/S checkpoint. In addition, it was shown that Bcr-Abl, the constitutively active, causative oncogene associated with some types of leukemia, drives cell proliferation by over-activating the two-step p27 phosphorylation of p27. NMR studies of phosphorylated p27 bound to Cdk2/cyclin A revealed the critical role that p27 flexibility plays in this two step phosphorylation mechanism, highlighting the role of intrinsic disorder in diverse regulatory function. We propose to marshal our significant expertise with p27 and Cdk2/cyclin A to extend our studies to other Cdk/cyclin complexes to comprehensively understand how p21 and p27 regulate cell division. These studies will not only provide insights into the fundamental mechanisms that control division in normal cells, but also will provide knowledge relevant to how these control mechanisms are corrupted in cancer cells. The four specific aims are: 1) To determine the effect of phosphorylation (of p27) on tyrosine 74 (Y74) by Src on the activity of p27 toward Cdk2/cyclin A. We will determine how Y74 phosphorylation, alone and in combination with tyrosine 88 (Y88) phosphorylation by Abl, alters the activity of p27 toward Cdk2/cyclin A using biochemical, biophysical and structural methods; 2) To determine the relevance of the two-step p27 phosphorylation/regulatory mechanism discovered for Cdk2/cyclin A to the regulation of other Cdk/cyclin complexes, including Cdk4/cyclin D1 and Cdk1/cyclin E2; 3) To determine the relevance of our discoveries concerning p27 phosphorylation to the related cell cycle regulator, p21; and 4) To investigate relationships between intrinsic flexibility and function for the different sub-domains of p27 and p21. [unreadable] [unreadable] [unreadable]